PROJECT SUMMARY Melanoma is the most lethal skin cancer and there remains a need to develop new therapies for patients with disseminated disease. By identifying the communication strategies cancer depends on to survive, the tumor microenvironment (TME) can serve as a fruitful area to identify new therapeutic targets. Melanoma cells interact with adipocytes within subcutaneous fat, but the consequences of this interaction is poorly understood. Our laboratory recently discovered that melanoma cells acquire lipids from stromal adipocytes and that this leads to increased melanoma proliferation and invasion. Lipid release from adipocytes is regulated by the breakdown of triglycerides into free fatty acids, a process known as lipolysis. The ability of cancer cells to induce adipocyte lipolysis has been observed, but the mechanism by which this occurs and the consequences for cancer progression remains unclear. Our preliminary data support that melanoma cells induce adipocyte lipolysis through a secreted factor. In AIM 1, we will investigate the mechanism of melanoma-induced adipocyte lipolysis using human melanoma cell lines and adipocytes in a co-culture system. We will first identify which signaling pathways are involved and then target candidate molecules that are known to mediate lipolysis through that pathway. As a byproduct of melanin synthesis, melanoma cells secrete catecholamines, which are the primary physiologic drivers of lipolysis. Therefore, we hypothesize that melanoma cells induce adipocyte lipolysis through secreted catecholamines. We will investigate the relationship between pigmentation in melanoma and adipocyte lipolysis to understand how differentiation programs influence interactions with the TME. In AIM2, we will examine the functional consequences of adipocyte lipolysis on melanoma progression using the zebrafish as an in vivo model. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for lipolysis and has been shown to be required for cancer-induced adipocyte lipolysis. We will create an adipocyte-restricted ATGL knockout in zebrafish, which offers the advantages of rapid transgenesis and high resolution imaging of cell-cell interactions in the TME. We will use this new in vivo model to determine the role of adipocyte lipolysis in melanoma initiation and metastasis. We hypothesize that blocking adipocyte lipolysis will decrease melanoma initiation and metastasis by cutting off access to extracellular lipids and creating an unfavorable microenvironment. By investigating the role of melanoma-induced adipocyte lipolysis, this proposal seeks to understand the contribution of adipocytes to melanoma progression and identify novel approaches to target the tumor microenvironment. !